DNA Barcoding Reveals Hidden Diatom Species in Hungary’s Vanishing Soda Lakes

In the shallow, hyperalkaline soda pans that dot the Carpathian Basin, the identity of a single microscopic alga can determine whether an entire lake is judged healthy or degraded. A new study published in Metabarcoding and Metagenomics has taken on one of the trickiest identification challenges in these extreme habitats: the diatom genus Halamphora, whose members are so similar under the microscope that even experts struggle to tell them apart. By pairing classical microscopy with DNA metabarcoding, researchers have now confirmed the genetic distinctness of a key salinity indicator species and uploaded its missing barcode to public reference databases, a small addition with large consequences for freshwater monitoring.

Diatoms, single-celled algae encased in ornate silica shells called frustules, are among the most trusted bioindicators in aquatic science. Under the European Water Framework Directive, benthic diatoms are one of five biological elements used to assess the ecological status of surface waters, with diatom indices built on the relative abundance of species and their known sensitivities to pollution and salinity. That system works only if species are identified accurately and consistently, which is precisely where the trouble begins. Traditional identification relies on subtle features of valve shape and stria density, demanding deep taxonomic expertise and often yielding subjective results.

DNA metabarcoding has been proposed as a more objective alternative. The technique amplifies short barcode regions of DNA from an environmental sample, sequences them in bulk on next-generation platforms, and matches the resulting sequences against reference databases to identify the taxa present. Yet studies comparing the two approaches have repeatedly found significant deviations, and the research team behind the new work points to one dominant cause: reference libraries are incomplete, particularly for species from unusual habitats. Soda pans, with their high sodium and hydrogen carbonate content, host rare and halophilic organisms that are exactly the kinds of species missing from genetic databases.

The loss of sodic character is the principal threat facing these habitats, as water management practices can channel lower-salinity water into the pans and dilute them. Accurate identification of species and precise knowledge of their salinity tolerance are therefore essential for judging ecological status. Within the framework of a Hungarian national monitoring project, the researchers sampled 37 soda pans and soda lakes across spring and autumn campaigns in 2019 and 2020, producing 69 samples analysed by light microscopy, of which 26 were also subjected to DNA sequencing. Twenty-two of the water bodies were subsaline and 15 hyposaline, ranging from natural to disturbed conditions.

The team focused on Halamphora, the fourth most common and seventh most species-rich genus among the 72 genera found in their soda samples. Two species in particular, Halamphora dominici and Halamphora veneta, occurred in nearly half of all samples and were frequently dominant, sometimes reaching relative abundances of 40 to 60 percent. The Halamphora veneta group, which also includes H. kevei and H. paraveneta, is notoriously difficult to resolve morphologically, with species boundaries resting on fine differences in stria density and valve outline that can shift with environmental stress, including salinity.

The molecular results delivered a striking revelation. Of 23 short amplicon sequence variants that automated pipelines assigned to H. veneta, phylogenetic analysis and BLAST searches showed they actually belonged to H. dominici. The culprit was a misidentified sequence in the Diat.barcode reference database. Using long-read sequencing of a sample where both species co-occurred, the researchers obtained a full 1547-nucleotide rbcL sequence for H. dominici that showed roughly 2 percent divergence from H. veneta, confirming the two are genuinely distinct species. The newly deposited sequences now supplement the reference library, and the team emphasizes that sequences should only enter databases after precise morpho-taxonomic verification.

Overall, six Halamphora species were detected in the soda habitats. Five were recovered by both methods, while H. oligotraphenta was found only by microscopy because it appeared exclusively in samples that were not sequenced. The comparison between techniques revealed a significant positive correlation between microscopy- and metabarcoding-based relative abundances for H. dominici, with a Pearson correlation coefficient of 0.73. Discrepancies in the other direction were also informative: DNA detected species in some samples where microscopy missed them, likely because sequencing draws on far more cells than the minimum 500 valves counted per slide, while some microscopically observed valves may have belonged to dead cells.

The ecological payoff is clear. Classified as mesohalobous, H. dominici showed a positive correlation with salinity, with an average salinity of 3.1 parts per thousand in the waters where it occurred, compared with 1.6 parts per thousand for the more cosmopolitan H. veneta, which tolerates freshwater to slightly brackish conditions. Given that the ecological status of Hungarian soda pans depends fundamentally on salinity, the correct discrimination between these two species, one a reliable flag of sodic character and the other a generalist, is critical for conservation-relevant assessments.

The study also contributed taxonomic housekeeping. By comparing length-to-width ratios and ultrastructural features, the researchers questioned whether Halamphora elongata is truly distinct from the older H. subcapitata, noting overlapping morphometrics and absent areolar occlusions in their specimens, though formal synonymisation awaits type-material analysis. Meanwhile, H. paraveneta, which lacks any reference sequence in Diat.barcode or GenBank, may have been lurking as an outlier sequence variant assigned to H. veneta, a hypothesis flagged for future work.

As environmental DNA methods face growing pressure to replace traditional microscopy in routine monitoring across Europe, this study offers both a caution and a roadmap. Misidentified dominant species in reference databases can silently distort the results of ecological status assessments, potentially misclassifying protected habitats. But the combination of meticulous morphology and DNA sequencing, applied to extreme habitats that represent environmental endpoints of genetic diversification, can close the gaps. For Hungary’s soda pans, some of Europe’s most distinctive and threatened aquatic ecosystems, that means the microscopic sentinels of their salty character can finally be named with confidence.

Subject of Research: Diatom biodiversity and salinity indicators in Hungarian soda pans assessed by microscopy and rbcL metabarcoding

Article Title:  Halamphora taxa in Hungarian soda pans and shallow soda lakes detected via metabarcoding and microscopic analyses

Article References: Ács, É., Bíró, T., Boros, E., Dobosy, P., Duleba, M., Földi, A., Kiss, K. T., Levkov, Z., Orgoványi, P., Szén, O. P., Trábert, Z., Vadkerti, E., & Grigorszky, I. (2023). Halamphora taxa in Hungarian soda pans and shallow soda lakes detected via metabarcoding and microscopic analyses. Metabarcoding and Metagenomics, 7, Article e111679. https://doi.org/10.3897/mbmg.7.111679

Image Credits: AI Generated

DOI: 10.3897/mbmg.7.111679

Keywords: diatoms, metabarcoding, soda pans, Halamphora, salinity, rbcL, ecological status, Water Framework Directive, reference database, Hungary, bioindicators, phylogeny